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Nano gold mark silver dyeing detection method of gene chip

A gene chip and nano-gold technology, which is applied in the field of nano-gold-labeled silver staining detection method, can solve the problems of expensive gene chip detection equipment, etc., and achieve the effects of improving the high price of the chip, reducing the use cost and having a long validity period.

Inactive Publication Date: 2003-01-22
刘全俊 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved in the present invention is to provide a nano-gold-labeled silver-stained gene chip detection technology that does not require expensive detection instruments and has a long effective storage time for the reagents, aiming at the problem that the current gene chip detection equipment is expensive

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015]Hepatitis B detection gene chip A gene chip nano-gold labeled silver staining detection method process: 1. Prepare the hepatitis B detection gene chip; 2. Use the SDS boiling method for gene extraction according to the reagents provided in the kit; take 20 μl Serum was added to the lysate, mixed evenly, boiled for 10 minutes, and centrifuged at 12,000 rpm for 10 minutes; 3. According to the gene fragments used in the chip, gene extraction and gene amplification were performed. The primers included with the chip kit were used, and the 5' end of one of each pair of primers was labeled with biotin, such as 5'-biotin-CCTGGTTATCGCTGGATG-3'; 5'-AGGGTTCAAATGTATACCC-3'. Carry out the PCR temperature cycle according to the conditions specified by the chip, first denaturation at 95°C for 3 minutes; then cycle 30 times at 95°C for 30 seconds, 52°C for 30 seconds, 72°C for 30 seconds, and finally, extend at 72°C for 4 minutes; 4. Take the blocking solution in the chip kit to block t...

Embodiment 2

[0017] The nano-gold-labeled silver staining detection process of various blood infectious disease detection gene chips: 1. Take the blood infectious disease detection gene chip, and the detection of various genes (such as HTLV, HIV, HBV, HCV, HPVS, EV, CMV, EBV, HSV) use polyoligonucleotides, the oligonucleotide sequence contains the conservative sequence of the detection target gene, and the detection objects include RNA pathogens and DNA pathogens; Extraction; note that the RNA should be prevented from being enzymatically hydrolyzed during the experiment; use RNase free equipment; 3. Take 10uL of the extracted RNA to an RNase free microcentrifuge tube, add 2uL RT primer, and act for 10 minutes at 70 °C to make Denature the RNA, then place it on ice; 4. Take out a new RT tube, add 38uL DEPC water, and place it on ice. Add all the solution in the RT tube to the RNA tube in step 3 within a few minutes; react at 42°C for 45 minutes, and then react at 70°C for 10 minutes to comp...

Embodiment 31

[0019] Implementation example 31, self-made hepatitis B mutation detection gene chip; all mutation sites are located in the three bases at the 3' end of the forward primer, most of which are located at the first base position at the 3' end, and the 5' end is modified 2. According to the reagents provided in the kit, use the SDS boiling method for gene extraction; take 20 μl of serum, add it to the lysate, mix well, boil for 10 minutes, and centrifuge at 12,000 rpm for 10 minutes 3. Carry out gene extraction and gene amplification according to the gene fragments used in the chip. Use 2×HotStar Tag RCR buffer, 50μM dNTPs, 20μg / μl BSA, 0.1-0.4uM reverse primer and 3u HotStar Tag DNA polymerase (Qiagen); the 5′ end of each reverse primer is labeled with biotin, such as 5'-biotin-GGGCATTTGGTGGTCT(G / A)T-3'; 5'-biotin-AGGGTTCAAATGTATA CCC-3'. Carry out the PCR temperature cycle according to the conditions specified by the chip, first denaturing at 95°C for 4 minutes; then cycle 35 t...

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Abstract

The present invention relates to gene chip detection method and is one using no radioactive label and no fluorescent label. The detection method includes the following steps: extracting the tested target gene in sample, labeling the gene with digoxin or biotin to obtain labeled DNA or RNA for hybridization with the gene chip; making nano gold labeled digoxin antibody to combined with digoxin or nano gold labeled avidin to combine with biotin; dyeing the hybridized gene chip with silver dyeing reagent; direct microscope oservation, CCD record of signal or scanning detection with common opticalscanning instrument to obtain corresponding crossing result; and further analysis by using relative software.

Description

technical field [0001] The invention relates to a nano-gold label silver staining detection method of a gene chip, which relates to a gene chip detection method, in particular to a non-radioactive label and non-fluorescence label detection method of the gene chip. Background technique [0002] DNA chip (gene chip) is one of the major scientific and technological developments with the most epochal characteristics in the high-tech field in recent years. It is a high-tech cross-combination of microelectronics, physics, chemistry, material science and life science. Gene chip [also known as DNA chip, biochip, DNA microprobe array (microarray) is to immobilize a large number of gene probes on a solid carrier according to a specific arrangement, and it performs complementary matching with the base sequence of the detected gene. , realizing the molecular recognition of nucleic acid sequences is an important means to efficiently obtain relevant biological information on a large scal...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 刘全俊赵伟刘伟陆祖宏朱纪军王宏
Owner 刘全俊
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